Adenosine deaminase (ADA) has been used in the treatment of an enzyme deficiency disorder called severe combined immunodeficiency disease (SCID) or “Bubble boy” disease for some time. For more than 15 years, Enzon Pharmaceuticals has made therapeutic ADA available for patients in the form of a PEGylated ADA prepared using a bovine source of the ADA enzyme.
Recently, there have been efforts to replace the bovine source enzyme with a recombinant source enzyme (hereinafter “rADA”). Both recombinant human (“rhADA”) and recombinant bovine (“rbADA”) have been considered as replacements for purified natural bovine ADA. The rbADA and rhADA enzymes are somewhat less stable than the native purified bovine enzyme that is currently employed. Both rhADA and rbADA are believed to degrade in a manner consistent with cysteine degradation: addition of oxygen; formation of dithiols; increasing degradation as pH increases; precipitation, especially as the pH is increased and the samples are concentrated. In the reduced state, cysteine contains a reactive —SH group (sulfhydryl) which is the form responsible for die degradation.
Evidence has suggested that a single, exposed cysteine may be responsible for the degradation that is seen for both rbADA and rhADA. Bovine ADA (i.e., native bovine ADA purified from bovine source) has a structure very similar to that of rhADA: both bovine ADA and rhADA have the same number of cysteines in the same positions of the primary sequence. Currently obtained recombinant human and recombinant bovine ADA contain degradants/impurities (dithiols) that are consistent with cysteine reactivity. Native bovine ADA differs structurally from recombinant bovine ADA in that native bovine ADA has a single mole of cysteine bound to each mole of ADA. Native bovine ADA is also stable to high pH, suggesting that the cysteine bound to the ADA is functioning as a protecting group.
One method for stabilizing recombinant human and/or recombinant bovine ADA is to cap the active Cys residue (Cys 74 of both mature rbADA and mature rhADA) with any one of oxidized glutathione, iodoacetamide, iodoacetic acid, cystine, other dithiols and mixtures thereof. This method is set forth by co-owned U.S. patent application Ser. No. 11/738,012, entitled, “Stabilized Proteins”, the contents of which are incorporated by reference herein in their entirety.
Despite the foregoing, it would be advantageous to avoid the need for an additional capping step by modifying the protein structure to provide inherent stability immediately upon expression. U.S. Pat. No. 5,346,823 describes the stabilization of prokaryotic proteases such as subtilisin, and neutral protease, by replacing destabilizing Cys residues with Ser and other amino acid residues, by mutation. However, mutational analysis of active sites in ADA revealed that replacement of a Cys residue (Cys 262) resulted in an enzyme with significantly decreased activity, Bhaumik et al. 1993, The J. of Biol Chem, 268. (8):5464-5470. Thus, before the present invention, it was not known to stabilize adenosine deaminase enzymes by replacing an active and exposed Cys residue by another amino acid residue while retaining optimal useful enzyme activity.
Thus, it would be beneficial to provide both rbADA and rhADA that is stable, i.e., without significant degradation during storage and processing, at pH levels which are useful for optimum PEGylation of the enzyme.